α-Amylase from wheat (Triticum aestivum) seeds: Its purification, biochemical attributes and active site studies

•Wheat α-amylase was purified to homogeneity and its identity confirmed by MALDI-TOF.•Biochemical characterisation revealed resistance to SDS denaturation at low concentrations.•High kcat objectifies wheat α-amylase for starch based and related industries.•Chemical modification studies showed histidine presence at the enzyme’s active site.

Glycosylated α-amylase from germinated wheat seeds (Triticum aestivum) has been purified to apparent electrophoretic homogeneity with a final specific activity of 1372 U/mg. The enzyme preparation when analysed on SDS–PAGE, displayed a single protein band with Mr 33 kDa; Superdex 200 column showed Mr of 32 kDa and MS/MS analysis further provided support for these values. The enzyme displayed its optimum catalytic activity at pH 5.0 and 68 °C with an activation energy of 6.66 kcal/mol and Q10 1.42. The primary substrate for this hydrolase appears to be starch with Km 1.56 mg/mL, Vmax 1666.67 U/mg and kcat 485 s−1 and hence is suitable for application in starch based industries. Thermal inactivation of α-amylase at 67 °C resulted in first-order kinetics with rate constant (k) 0.0086 min−1 and t1/2 80 min. The enzyme was susceptible to EDTA (10 mM) with irreversible loss of hydrolytic power. In the presence of 1.0 mM SDS, the enzyme lost only 14% and 23% activity in 24 and 48 h, respectively. Chemical modification studies showed that the enzyme contains histidine and carboxylic residues at its active site for its catalytic activity and possibly conserved areas.